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Single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe for the detection of rare mutations

Abstract

The detection of rare mutant DNA from a background of wild-type alleles usually requires laborious manipulations, such as restriction enzyme digestion and gel electrophoresis. Here, we describe a protocol for homogeneous detection of rare mutant DNA in a single tube. The protocol uses a peptide nucleic acid (PNA) as both PCR clamp and sensor probe. The PNA probe binds tightly to perfectly matched wild-type DNA template but not to mismatched mutant DNA sequences, which specifically inhibits the PCR amplification of wild-type alleles without interfering with the amplification of mutant DNA. A fluorescein tag (which undergoes fluorescence resonance energy transfer with the adjacent fluorophore of an anchor probe when both are annealed to the template DNA) also allows the PNA probe to generate unambiguous melting curves to detect mutant DNA during real-time fluorescent monitoring. The whole assay takes about only 1 h. This protocol has been used for detecting mutant K-ras DNA and could be applied to the detection of other rare mutant DNAs.

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Figure 1: Schematic diagram of the role of the PNA probe.
Figure 2: Probe and primer design.
Figure 3: Amplification curves of control PCR reactions under (a) non-clamping or (b) clamping conditions.
Figure 4: Determination of PCR efficiency as well as template quality through the amplification curves
Figure 5: Typical melting curves
Figure 6: Flowchart detailing the stages involved in the optimization and use of this single-tube assay for rare mutation detection using a PNA probe.

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References

  1. Iinuma, H. et al. Detection of tumor cells in blood using CD45 magnetic cell separation followed by nested mutant allele-specific amplification of p53 and K-ras genes in patients with colorectal cancer. Int. J. Cancer 89, 337–344 (2000).

    Article  CAS  Google Scholar 

  2. Nickerson, D.A. et al. Automated DNA diagnostics using an ELISA-based oligonucleotide ligation assay. Proc. Natl. Acad. Sci. USA 87, 8923–8927 (1990).

    Article  CAS  Google Scholar 

  3. Dieterle, C.P., Conzelmann, M., Linnemann, U. & Berger, M.R. Detection of isolated tumor cells by polymerase chain reaction-restriction fragment length polymorphism for K-ras mutations in tissue samples of 199 colorectal cancer patients. Clin. Cancer Res. 10, 641–650 (2004).

    Article  CAS  Google Scholar 

  4. Jacobson, D.R. & Mills, N.E. A highly sensitive assay for mutant ras genes and its application to the study of presentation and relapse genotypes in acute leukemia. Oncogene 9, 553–563 (1994).

    CAS  PubMed  Google Scholar 

  5. Norheim Andersen, S. et al. K-ras mutations and HLA-DR expression in large bowel adenomas. Br. J. Cancer 74, 99–108 (1996).

    Article  CAS  Google Scholar 

  6. Imai, M., Hoshi, T. & Ogawa, K. K-ras codon 12 mutations in biliary tract tumors detected by polymerase chain reaction denaturing gradient gel electrophoresis. Cancer 73, 2727–2733 (1994).

    Article  CAS  Google Scholar 

  7. Nishikawa, T. et al. A simple method of detecting K-ras point mutations in stool samples for colorectal cancer screening using one-step polymerase chain reaction/restriction fragment length polymorphism analysis. Clin. Chim. Acta 318, 107–112 (2002).

    Article  CAS  Google Scholar 

  8. Toyooka, S. et al. Detection of codon 61 point mutations of the K-ras gene in lung and colorectal cancers by enriched PCR. Oncol. Rep. 10, 1455–1459 (2003).

    CAS  PubMed  Google Scholar 

  9. Maekawa, M. et al. Three-dimensional microarray compared with PCR-single-strand conformation polymorphism analysis/DNA sequencing for mutation analysis of K-ras codons 12 and 13. Clin. Chem. 50, 1322–1327 (2004).

    Article  CAS  Google Scholar 

  10. Lleonart, M.E., Ramon y Cajal, S., Groopman, J.D. & Friesen, M.D. Sensitive and specific detection of K-ras mutations in colon tumors by short oligonucleotide mass analysis. Nucleic Acids Res.. 32, e53 (2004).

    Article  Google Scholar 

  11. Sun, X., Hung, K., Wu, L., Sidransky, D. & Guo, B. Detection of tumor mutations in the presence of excess amounts of normal DNA. Nat. Biotechnol. 20, 186–189 (2002).

    Article  CAS  Google Scholar 

  12. Lilleberg, S.L., Durocher, J., Sanders, C., Walters, K. & Culver, K. High sensitivity scanning of colorectal tumors and matched plasma DNA for mutations in APC, TP53, K-RAS, and BRAF genes with a novel DHPLC fluorescence detection platform. Ann. NY Acad. Sci. 1022, 250–256 (2004).

    Article  CAS  Google Scholar 

  13. Luo, J.D. et al. Detection of rare mutant K-ras DNA in a single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe. Nucleic Acids Res. 34, e12 (2006).

    Article  Google Scholar 

  14. Lay, M.J. & Wittwer, C.T. Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. Clin. Chem. 43, 2262–2267 (1997).

    CAS  PubMed  Google Scholar 

  15. Bernard, P.S., Ajioka, R.S., Kushner, J.P. & Wittwer, C.T. Homogeneous multiplex genotyping of hemochromatosis mutations with fluorescent hybridization probes. Am. J. Pathol. 153, 1055–1061 (1998).

    Article  CAS  Google Scholar 

  16. Rodriguez-Manotas, M. et al. Real time PCR assay with fluorescent hybridization probes for genotyping intronic polymorphism in presenilin-1 gene. Clin. Chim. Acta 364, 343–344 (2006).

    Article  CAS  Google Scholar 

  17. Heesen, M., Wessiepe, M., Kunz, D., Vasickova, K. & Blomeke, B. Rapid and reliable genotyping for the Toll-like receptor 4 A896G polymorphism using fluorescence-labeled hybridization probes in a real-time polymerase chain reaction assay. Clin. Chim. Acta 333, 47–49 (2003).

    Article  CAS  Google Scholar 

  18. Popp, J., Messner, B. & Steimer, W. High-speed genotyping of CYP1A2*1F mutation with fluorescent hybridization probes using the LightCycler. Pharmacogenomics 4, 643–646 (2003).

    Article  CAS  Google Scholar 

  19. Egholm, M. et al. PNA hybridizes to complementary oligonucleotides obeying the Watson–Crick hydrogen-bonding rules. Nature 365, 566–568 (1993).

    Article  CAS  Google Scholar 

  20. Hanvey, J.C. et al. Antisense and antigene properties of peptide nucleic acids. Science 258, 1481–1485 (1992).

    Article  CAS  Google Scholar 

  21. Nielsen, P.E., Egholm, M., Berg, R.H. & Buchardt, O. Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, 1497–1500 (1991).

    Article  CAS  Google Scholar 

  22. Orum, H. et al. Single base pair mutation analysis by PNA directed PCR clamping. Nucleic Acids Res. 21, 5332–5336 (1993).

    Article  CAS  Google Scholar 

  23. Taback, B. et al. Peptide nucleic acid clamp PCR: a novel K-ras mutation detection assay for colorectal cancer micrometastases in lymph nodes. Int. J. Cancer 111, 409–414 (2004).

    Article  CAS  Google Scholar 

  24. Hancock, D.K., Schwarz, F.P., Song, F., Wong, L.J. & Levin, B.C. Design and use of a peptide nucleic acid for detection of the heteroplasmic low-frequency mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) mutation in human mitochondrial DNA. Clin. Chem. 48, 2155–2163 (2002).

    CAS  PubMed  Google Scholar 

  25. Takiya, T. et al. Identification of single base-pair mutation on uidA gene of Escherichia coli O157:H7 by peptide nucleic acids (PNA) mediated PCR clamping. Biosci. Biotechnol. Biochem. 68, 360–368 (2004).

    Article  CAS  Google Scholar 

  26. Kirishima, T. et al. Detection of YMDD mutant using a novel sensitive method in chronic liver disease type B patients before and during lamivudine treatment. J. Hepatol. 37, 259–265 (2002).

    Article  CAS  Google Scholar 

  27. Ohishi, W. et al. Identification of rare polymerase variants of hepatitis B virus using a two-stage PCR with peptide nucleic acid clamping. J. Med. Virol. 72, 558–565 (2004).

    Article  CAS  Google Scholar 

  28. Kreuzer, K.A. et al. Preexistence and evolution of imatinib mesylate-resistant clones in chronic myelogenous leukemia detected by a PNA-based PCR clamping technique. Ann. Hematol. 82, 284–289 (2003).

    Article  CAS  Google Scholar 

  29. Chen, C.Y., Shiesh, S.C. & Wu, S.J. Rapid detection of K-ras mutations in bile by peptide nucleic acid-mediated PCR clamping and melting curve analysis: comparison with restriction fragment length polymorphism analysis. Clin. Chem. 50, 481–489 (2004).

    Article  CAS  Google Scholar 

  30. Dabritz, J., Hanfler, J., Preston, R., Stieler, J. & Oettle, H. Detection of Ki-ras mutations in tissue and plasma samples of patients with pancreatic cancer using PNA-mediated PCR clamping and hybridisation probes. Br. J. Cancer 92, 405–412 (2005).

    Article  CAS  Google Scholar 

  31. Nagai, Y. et al. Genetic heterogeneity of the epidermal growth factor receptor in non-small cell lung cancer cell lines revealed by a rapid and sensitive detection system, the peptide nucleic acid-locked nucleic acid PCR clamp. Cancer Res. 65, 7276–7282 (2005).

    Article  CAS  Google Scholar 

  32. Eckert, K.A. & Kunkel, T.A. DNA polymerase fidelity and the polymerase chain reaction. PCR Methods Appl. 1, 17–24 (1991).

    Article  CAS  Google Scholar 

  33. Braasch, D.A. & Corey, D.R. Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA. Chem. Biol. 8, 1–7 (2001).

    Article  CAS  Google Scholar 

  34. Kutyavin, I.V. et al. 3′-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic. Acids Res. 28, 655–661 (2000).

    Article  CAS  Google Scholar 

  35. Jahr, S. et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 61, 1659–1665 (2001).

    CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by Chang Gung Molecular Medicine Research Center and grants from Chang Gung Memorial Hospital (CMRPD140041), Ministry of Education (EMRPD 15027), and National Science Council, Taiwan, ROC (NSC 92-2622-B-182-001-CC3).

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Correspondence to Chiuan-Chian Chiou.

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The Chang Gung University will file a patent related to this method.

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Chiou, CC., Luo, JD. & Chen, TL. Single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe for the detection of rare mutations. Nat Protoc 1, 2604–2612 (2006). https://doi.org/10.1038/nprot.2006.428

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